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. Author manuscript; available in PMC: 2020 Feb 1.
Published in final edited form as: Nitric Oxide. 2018 Dec 24;83:51–64. doi: 10.1016/j.niox.2018.12.007

Figure 4. Opposing roles of GSNO vs. ONOO− in thrombin-induced cell signaling for endothelial barrier disruption in hBMVECs.

Figure 4.

Human brain microvessel endothelial cells (hBMVECs) were treated with various concentrations of GSNO or SIN-1 (ONOO− donor), incubated for 2hr, and cellular levels of S-nitrosylated proteins and RhoA (A-i) and tyrosine-nitrated proteins and RhoA (A-ii) were analyzed as described in method section. hBMVECs were treated with thrombin (0.1 unit/ml for 5min), in the presence or absence of various concentrations GSNO or SIN-1 (pretreated for 2hr), and RhoA activity was analyzed as described in method section (B). hBMVECs were treated with thrombin (0.1 unit/ml) in the presence or absence of various concentrations GSNO or SIN-1 and intracellular Ca2+ ([Ca2+]i) influx (C-i and ii) and cell viability (MTT assay) (C-iii) were analyzed. hBMVECs were treated with thrombin (0.1 unit/ml for 5min), in the presence or absence of various concentrations GSNO or SIN-1 (D-i) or decomposed GSNO (100µM) or SIN-1 (1000µM) (D-ii), and MLC phosphorylation was analyzed by Western analysis. β-actin was used for internal loading control for Western analysis. The vertical bars are means of individual data and T-bars are standard deviation. *** p ≤ 0.001 as compared to the control group. + p ≤ 0.05 and ++ p ≤ 0.01 as compared to thrombin treated group. All experiments were repeated at least three times.